Understanding the biology of individual cells within complex microenvironments has proven to be a particular challenge despite the increasing number and variety of tools available for the analysis of single cells at the cellular and molecular levels, including microscopy, polymerase chain reaction (PCR), patch-clamp, and microanalytical chemical separations. One obstacle to being able to gain this understanding is the limited set of tools that permit the selection of a single cell of defined phenotype from a large cellular population, for example, fluorescence-activated cell sorting (FACS) or laser capture microdissection. The repertoire of available tools is further diminished when one is interested in recovering a single adherent cell from culture with minimal cellular perturbation, that is, without the need to strip the cell from the growth surface such as is required with popular cell-sorting technologies such as FACS or if maintenance of cellular viability is important.
A microtechnology for the isolation and collection of single adherent cells from large heterogeneous populations has been previously reported. (See Wang, Y.; Sims, C. E.; Marc, P.; Bachman, M.; Li, G. P.; Allbritton, N. L. Langmuir 2006, 22, 8257-8262; To'a Salazar, G.; Wang, Y.; Young, G.; Bachman, M.; Sims, C. E.; Li, G. P.; Allbritton, N. L. Anal. Chem. 2007, 79, 682-687; Wang, Y.; Young, G.; Bachman, M.; Sims, C. E.; Li, G. P.; Allbritton, N. L. Anal. Chem. 2007, 79, 2359-2366; and Wang, Y.; Young, G.; Aoto, P. C.; Pai, J.; Bachman, M.; Li, G. P.; Sims, C. E.; Allbritton, N. L. Cytometry, Part A 2007, 71A, 866-874.) This “micropallet array” technology is a unique cell-handling platform that comprises hundreds of thousands of microscale pedestals (“micropallets”). Micropallet arrays are constructed using standard photolithographic techniques from high aspect ratio photoresist, and the technology was developed using the popular epoxy-based negative photoresist SU-8.
A new high aspect ratio negative photoresist, called “1002F photoresist”, for micropallet array fabrication has also been reported. (See Pai, J.; Wang, Y.; Salazar, G. T.; Sims, C. E.; Bachman, M.; Li, G. P.; Allbritton, N. L. Anal. Chem. 2007, 79, 8774-8780.) 1002F photoresist is similar to SU-8 but offers improved biophysical properties over SU-8, specifically markedly lower autofluorescence across a broad range of UV and visible light wavelengths, and increased biocompatibility and support for cell adhesion as compared with SU-8. (Id.)
In the current state of the technology, individual micropallets that are released from the micropallet array are transferred to collection wells by inverting the micropallet array over a large collection area or array of collection wells. The released micropallets (free floating) fall into the collection well(s) via the action of gravity, while unreleased micropallets remained affixed to the glass substrate. The disadvantages to this method are several fold: 1) Generally only one micropallet can be released and collected in each inversion process (due to confusion of micropallet identities if multiple, un-indexed micropallets are simultaneously released and collected); 2) In the inversion method, the array of collection wells must be scanned to find the released micropallet. These two disadvantages dramatically reduce the achievable throughput of the system; 3) By nature of this method, the collected micropallet is present within a relatively very large volume of liquid after collection. While appropriate for clonal expansion of the collected cell, for single cell PCR analysis of the cell's DNA or RNA, the cell must first be moved to a much smaller volume of liquid.
Therefore, an improved micropallet that facilitates collection and/or manipulation is desirable.